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Megan Gragg, Paul Park; Structural and Phenotypic Characterization of a Completely Misfolded Rhodopsin Mutant. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4546.
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© ARVO (1962-2015); The Authors (2016-present)
A majority of rhodopsin-mediated autosomal dominant retinitis pigmentosa (adRP) has been linked to mutations resulting in misfolded rhodopsin aggregates. This includes the most common rhodopsin mutation in the United States, the P23H mutation. The mechanism of rhodopsin-mediated adRP is unclear. The extensively studied P23H mutation causes partial misfolding of rhodopsin, which results in a combination of properly folded and aggregated misfolded receptor. The multiple receptor species both can contribute to the disease, and therefore the underlying disease mechanism is ambiguous. To characterize the structure and effect of misfolded rhodopsin aggregates alone and to better understand the mechanism of adRP, the completely misfolded G188R rhodopsin mutant was investigated.
To structurally characterize misfolded rhodopsin aggregates, Förster Resonance Energy Transfer (FRET) was used to monitor interactions between fluorescently tagged rhodopsins expressed in transfected HEK293 cells. We developed a FRET method that distinguishes between properly folded oligomers and misfolded aggregates of rhodopsin. To examine the effect of misfolded rhodopsin mutants on the retina, a mouse model expressing G188R rhodopsin was generated by CRISPR/Cas9. Retinal degeneration was characterized by histology and compared to results from C57Bl/6J mice.
G188R mutant rhodopsin expressed in HEK293 cells only formed aggregates and treatment with 9-cis retinal did not prevent receptor misfolding and aggregation. To examine the effect of misfolded rhodopsin aggregates on the retina, G188R rhodopsin heterozygous mice were examined. At four weeks of age, the G188R rhodopsin heterozygous mice had less nuclei in the outer nuclear layer of the retina than in aged matched C57Bl/6J mice. By six months of age, no nuclei remained in the outer nuclear layer of G188R heterozygous mice.
The G188R mutation in rhodopsin causes misfolding and aggregation of the receptor. Aggregates of misfolded receptor results in progressive retinal degeneration in mice that is complete by six months of age. Aggregation of G188R rhodopsin cannot be prevented by chromophore supplementation, and therefore this previously proposed therapeutic strategy is predicted to be ineffective.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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